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In situ effects of S1 endonuclease (Neurospora crassa) on pachytene chromatin of Xenopus laevis ♀ at metamorphosis
Preliminary notes
439
be single-stranded to a large extent and, therefore, stain purple red with Unna [5,6]. A recent electron microscopic analysis of Xenopus amplified rDNA by BuongiomoNardelli et al. [7] supports this suggestion. Besides already described circular DNA References molecules and “rolling circles” [8, 93, the 1. Baker, J B & Humphreys, T, Science 175 (1972) authors have observed partially single905. 2. Borek, C, Grob, M &Burger, M M, Exp cell res 77 stranded forms, very similar to the “D” (1973) 207. loops and gapped circles found in DNA. 3. Burger, M M, Fed proc 32 (1973) 91. 4. Elligsen, J D, Thompson, J E & Frey, H E, Exp Linear molecules containing conspicuous cell res 92 (1975) 87. single-stranded regions, which are difficult 5. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1%9) 1418. to interpret, were also found. 6. Kaplan, J & Moskowitz, M, Biochim biophys acta We have approached the problem in quite 389 (1975) 306. 7. Smets, LA, DeLey, L & Collard, J G, Exp cell res a different way, following the effects of an 95 (1975) 95. endonuclease, specific for single-stranded 8. Sutherland, R M, McCredie, J A & Inch, W R, J natl cancer inst 46 (1971) 113. DNA, on fixed sections of young Xenopus 9. Thompson, J E, Elligsen, J D & Frey, H E, J cell ovaries. We have compared the behaviour sci 18 (1975) 427. of “cap” chromatin and pachytene chromoReceived September 17, 1976 somal chromatin towards this enzyme, as Accepted October 15, 1976 well as that of somatic nuclei (follicular cells). deed it has recently been observed that even transformed cells exhibit higher agglutinabilities at confluence than at subconfluence [7].
Material and Methods In situ effects of Sl endonuclease (Neurospora crassa) on pachytene chromatin of Xempus Levis 9 at metamorphosis A. FICQ, Department of Molecular sity of Brussels, Belgium
Biology, Univer-
The genes coding for rRNA inxenopus laevis females are amplified at early meiosis and are localized in a “cap” overlying the condensed pachytene chromosomes [ 1, 2, 31. The “cap” stains red and the pachytene chromosomes green, after Unna staining (methyl green-pyronin) [4]; this suggests that the “cap” and the chromosomal chromatin display different physico-chemical features probably related to the amplification process. Among the hypotheses proposed to explain this difference, we have suggested that the amplified rDNA of the “cap” might 29-761813
For each experiment (9 in total) 2&tO ovaries of Xenopus at metamorphosis were dissected, fixed by freeze-substitution or- acetic+lcohol (1: 3) embedded and sectioned at 7 Nrn on gelatinized slides. Prior to this, the dissected ovaries had been incubated for 24 h, at 6°C in 3 ml of TC 199 modified Difco Medium [lo] supplemented with 100&i [3H]TdR (The Radiochemical Centre, Amersham, Bucks, UK). The methods of Dreskin & Mayall [ 1l] were used for the following treatments. Several sections were also submitted to alkaline denaturation [12]. The first assay was carried out with Sl endonuclease of Neurospora crassa (a generous gift of Dr A. Falaschi). For the following in situ experiments, we have used Neurospora crassa endonuclease (EC 3.1.4.21. Boehringer, spec. act. 400-800 units/n@. Enzyme activity and specificity were kindly tested by Dr J. Rommelaere. Digestion with Sl endonuclease (0.2 or 2.5 units/ml in Tris-HCl buffer, 0.1 units/ml in MgCIZ, 0.01 M, pH 8.1) was performed at 37°C for 30 mitt, 2 or 3 h, in humid chambers, as described previously [13] and autoradiographed [ 141.(Exposure time: 8 days, for the counting; 15 days for the micrographs.) Control slides treated with RNAase alone, some of which had been submitted to thermal denaturation without Sl endonuclease treatment, were also prepared and autoradiographed. In addition, some slides were treated with pancreatic DNAase (Worthington, 0.1 mg/ml, MgCIZ 300 M, 30 min at 37°C). Exp Cdl Res la4 (1977)
and the intensity of labelling with [3H]TdR, after various experimental treatments. Thermal denaturation modifies Unna staining: all the cellular structures, including the nuclei, stain red; this demonstrates that DNA denaturation took place in situ at 100°C. Thermal denaturation followed by Sl endonuclease digestion affects the cell morphology. In particular, the pachytene “caps” no longer present their compact structure (fig. 3). After endonuclease digestion alone, without previous RNAase treatment and denaturation, the “caps” stain bluish with Unna. After alkaline denaturation an analysis of ovary sections was not possible, since the cellular structures were no longer identifiable with certainty. Tables 1 and 2 present the numerical data for silver grains counting on the autoradiographs in the different experimental conditions. After treatment at 20 and at 6O”C, i.e. under non-denaturing conditions, Sl endonuclease affects only the labelling of the pachytene “caps” without affecting that Table 1. Average number of Ag grains/ pachytene “cap” [3H]TdR, 100 &i/3 ml. 50 pachytene cells were counted in each case
Fig. I. Autoradiograph of young Xenopus ovary sections. The somatic nuclei and the pachytene “caps” are labelled wim LJH]TdR. Controls. x800. Fig. 2. Autoradiograph of young Xenopus ovary sections after Sl endonuclease treatment without previous thermal denaturation (2.5 units/ml for 3 h). The somatic nuclei remain labelled but the labelling of the “caps” is, now, very low. x800. Fig. 3. Autoradiograph of young Xenopus ovary sections after thermal denaturation (100°C) followed by S 1 endonuclease digestion. Radioactivity is greatly decreased or absent in both somatic nuclei and “caps” x800.
Results The micrographs (figs 1, 2, 3) demonstrate the morphological state of the ovarian cells Exp CellRes 104 (1977)
Controls Buffer controls RNAase treatment without endonuclease RNAase treatment then treatment at WC (10 min) without endonuclease Endonuclease (37°C) 0.2 units/nil after RNAase Endonuclease (37°C) 2.5 units/ml after RNAase Endonuclease (37°C) after RNAase and denaturation at 100°C Pancreatic DNAase (30 mitt)
10.5 12.4 9.8
11.5 30min 9
2h 5.3
3h 3.1
30min 0.8
2h 0.9
3h 0.7
30min
2h
3h
0
0
0
0
-
-
Preliminary notes Table 2. Percentage of labelled pachytene “caps” [3H]TdR 100 &i/3 ml. 50 pachytene cells were counted in each case Controls Buffer controls RNAase treatment without endonuclease RNAase treatment then 60°C (10 min) without endonuclease Endonuclease (37°C) 0.2 units/ml after RNAase Endonuclease (37°C) 2.5 units/ml after RNAase Endonuclease (37°C) after RNAase and denaturation at 1OtX) Pancreatic DNAase (30 min)
70% 73% 62% 59.8 % 30 min 36%
4%
3h 13%
30min 32%
2h 12%
3h 3%
30min
2h
3h
0
0
0
0
--
of the follicular cell nuclei (table 2 is justified by the fact that all the pachytene“caps” are not at the same stage of development). After thermal denaturation at lOO“C, the follicular cell nuclei, which were previously labelled, are also affected by the enzyme (fig. 3). After pancreatic DNAase digestion, radioactivity can no longer be observed. Discussion It is logical to think that the presence of proteins in the chromatin of ovary sections strongly decreases the accessibility of Sl endonuclease to DNA; this explains why one must use prolonged times of incubation and high concentrations of the enzyme in order to follow its effects in situ, as we have tried to do. Although the morphology of the “caps” which are very compact structures, does not seem affected by the enzymic treatment, without previous thermal denaturation, the [3H]TdR labelled rDNA which they contain is hydrolysed at a temperature (37°C) where Sl endonuclease is
441
almost ineffective upon double-stranded DNA. Its action is detectable on doublestranded DNA only after thermal denaturation at 100°C [ll]. Under such conditions (after heating at lOO”C), besides “cap” DNA, the follicular cell nuclei are affected by the enzyme. Our results, in this respect, confirm those of Dreskin & Mayall [ 1l] who studied the effects of thermal denaturation on human leukocyte chromatin in situ with a different staining method (gallocyanin chromalun). Besides the fact that rapid replication takes place in rDNA, our present in situ observations on the effects of Sl endonuclease from Neurospora crassa confir~-~~ that single-stranded forms are present in “cap” DNA, that they are labelled with r3H]TdR and that they could play an intermediary role in the gene amplification process. We wish to thank Dr Christopher Evans for kind help in the preparation of the manuscript.
References 1. Gall,JG&Pardue,ML,ProcnatlacadsciUS63 (1%9) 378. 2. MacGregor, H C, J cell sci 3 (1%8) 437. 3. Ficq, A, Exp cell res 53 (1968) 691. 4. -Ibid 63 (1970) 453. 5. Durand, M, Compt rend acad sci 234 (1952) 1579. 6. Thomas, R, Arch int physio161 (1953) 370. 7. Buongiomo-Nardelli, M, Amaldi, F & LavaSanchez, PA, Exp cell res 98 (1976) 95. 8. Hourcade, D, Dressler, D & Wolfson, J, Proc natl acad sci US 70 (1973) 2926. 9. Rochaix, J B, Bird, A & Bakken, A, J mol biol 87 (1974) 473. 10. Gall, J G, Symposium on the nucleolus. Natl cancer inst monogr 23 (1966) 475. 11. Dreskin, S C & Mayall, B H, J histochem cyto-. them 22 (1974) 120. 12. Gall. J G. Proc natl acad sci US 60 (1968) 533. 13. Ficq; A, J microsc biol cell 25 (1976) 7. ’ 14. - The cell (ed J Brachet & A Mirsky) vol. I, p. 67. Academic Press, New York (1959). Received September 21, 1976 Accepted October 21, 1976
Exp Cell Res 104 (1977)
439
be single-stranded to a large extent and, therefore, stain purple red with Unna [5,6]. A recent electron microscopic analysis of Xenopus amplified rDNA by BuongiomoNardelli et al. [7] supports this suggestion. Besides already described circular DNA References molecules and “rolling circles” [8, 93, the 1. Baker, J B & Humphreys, T, Science 175 (1972) authors have observed partially single905. 2. Borek, C, Grob, M &Burger, M M, Exp cell res 77 stranded forms, very similar to the “D” (1973) 207. loops and gapped circles found in DNA. 3. Burger, M M, Fed proc 32 (1973) 91. 4. Elligsen, J D, Thompson, J E & Frey, H E, Exp Linear molecules containing conspicuous cell res 92 (1975) 87. single-stranded regions, which are difficult 5. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1%9) 1418. to interpret, were also found. 6. Kaplan, J & Moskowitz, M, Biochim biophys acta We have approached the problem in quite 389 (1975) 306. 7. Smets, LA, DeLey, L & Collard, J G, Exp cell res a different way, following the effects of an 95 (1975) 95. endonuclease, specific for single-stranded 8. Sutherland, R M, McCredie, J A & Inch, W R, J natl cancer inst 46 (1971) 113. DNA, on fixed sections of young Xenopus 9. Thompson, J E, Elligsen, J D & Frey, H E, J cell ovaries. We have compared the behaviour sci 18 (1975) 427. of “cap” chromatin and pachytene chromoReceived September 17, 1976 somal chromatin towards this enzyme, as Accepted October 15, 1976 well as that of somatic nuclei (follicular cells). deed it has recently been observed that even transformed cells exhibit higher agglutinabilities at confluence than at subconfluence [7].
Material and Methods In situ effects of Sl endonuclease (Neurospora crassa) on pachytene chromatin of Xempus Levis 9 at metamorphosis A. FICQ, Department of Molecular sity of Brussels, Belgium
Biology, Univer-
The genes coding for rRNA inxenopus laevis females are amplified at early meiosis and are localized in a “cap” overlying the condensed pachytene chromosomes [ 1, 2, 31. The “cap” stains red and the pachytene chromosomes green, after Unna staining (methyl green-pyronin) [4]; this suggests that the “cap” and the chromosomal chromatin display different physico-chemical features probably related to the amplification process. Among the hypotheses proposed to explain this difference, we have suggested that the amplified rDNA of the “cap” might 29-761813
For each experiment (9 in total) 2&tO ovaries of Xenopus at metamorphosis were dissected, fixed by freeze-substitution or- acetic+lcohol (1: 3) embedded and sectioned at 7 Nrn on gelatinized slides. Prior to this, the dissected ovaries had been incubated for 24 h, at 6°C in 3 ml of TC 199 modified Difco Medium [lo] supplemented with 100&i [3H]TdR (The Radiochemical Centre, Amersham, Bucks, UK). The methods of Dreskin & Mayall [ 1l] were used for the following treatments. Several sections were also submitted to alkaline denaturation [12]. The first assay was carried out with Sl endonuclease of Neurospora crassa (a generous gift of Dr A. Falaschi). For the following in situ experiments, we have used Neurospora crassa endonuclease (EC 3.1.4.21. Boehringer, spec. act. 400-800 units/n@. Enzyme activity and specificity were kindly tested by Dr J. Rommelaere. Digestion with Sl endonuclease (0.2 or 2.5 units/ml in Tris-HCl buffer, 0.1 units/ml in MgCIZ, 0.01 M, pH 8.1) was performed at 37°C for 30 mitt, 2 or 3 h, in humid chambers, as described previously [13] and autoradiographed [ 141.(Exposure time: 8 days, for the counting; 15 days for the micrographs.) Control slides treated with RNAase alone, some of which had been submitted to thermal denaturation without Sl endonuclease treatment, were also prepared and autoradiographed. In addition, some slides were treated with pancreatic DNAase (Worthington, 0.1 mg/ml, MgCIZ 300 M, 30 min at 37°C). Exp Cdl Res la4 (1977)
and the intensity of labelling with [3H]TdR, after various experimental treatments. Thermal denaturation modifies Unna staining: all the cellular structures, including the nuclei, stain red; this demonstrates that DNA denaturation took place in situ at 100°C. Thermal denaturation followed by Sl endonuclease digestion affects the cell morphology. In particular, the pachytene “caps” no longer present their compact structure (fig. 3). After endonuclease digestion alone, without previous RNAase treatment and denaturation, the “caps” stain bluish with Unna. After alkaline denaturation an analysis of ovary sections was not possible, since the cellular structures were no longer identifiable with certainty. Tables 1 and 2 present the numerical data for silver grains counting on the autoradiographs in the different experimental conditions. After treatment at 20 and at 6O”C, i.e. under non-denaturing conditions, Sl endonuclease affects only the labelling of the pachytene “caps” without affecting that Table 1. Average number of Ag grains/ pachytene “cap” [3H]TdR, 100 &i/3 ml. 50 pachytene cells were counted in each case
Fig. I. Autoradiograph of young Xenopus ovary sections. The somatic nuclei and the pachytene “caps” are labelled wim LJH]TdR. Controls. x800. Fig. 2. Autoradiograph of young Xenopus ovary sections after Sl endonuclease treatment without previous thermal denaturation (2.5 units/ml for 3 h). The somatic nuclei remain labelled but the labelling of the “caps” is, now, very low. x800. Fig. 3. Autoradiograph of young Xenopus ovary sections after thermal denaturation (100°C) followed by S 1 endonuclease digestion. Radioactivity is greatly decreased or absent in both somatic nuclei and “caps” x800.
Results The micrographs (figs 1, 2, 3) demonstrate the morphological state of the ovarian cells Exp CellRes 104 (1977)
Controls Buffer controls RNAase treatment without endonuclease RNAase treatment then treatment at WC (10 min) without endonuclease Endonuclease (37°C) 0.2 units/nil after RNAase Endonuclease (37°C) 2.5 units/ml after RNAase Endonuclease (37°C) after RNAase and denaturation at 100°C Pancreatic DNAase (30 mitt)
10.5 12.4 9.8
11.5 30min 9
2h 5.3
3h 3.1
30min 0.8
2h 0.9
3h 0.7
30min
2h
3h
0
0
0
0
-
-
Preliminary notes Table 2. Percentage of labelled pachytene “caps” [3H]TdR 100 &i/3 ml. 50 pachytene cells were counted in each case Controls Buffer controls RNAase treatment without endonuclease RNAase treatment then 60°C (10 min) without endonuclease Endonuclease (37°C) 0.2 units/ml after RNAase Endonuclease (37°C) 2.5 units/ml after RNAase Endonuclease (37°C) after RNAase and denaturation at 1OtX) Pancreatic DNAase (30 min)
70% 73% 62% 59.8 % 30 min 36%
4%
3h 13%
30min 32%
2h 12%
3h 3%
30min
2h
3h
0
0
0
0
--
of the follicular cell nuclei (table 2 is justified by the fact that all the pachytene“caps” are not at the same stage of development). After thermal denaturation at lOO“C, the follicular cell nuclei, which were previously labelled, are also affected by the enzyme (fig. 3). After pancreatic DNAase digestion, radioactivity can no longer be observed. Discussion It is logical to think that the presence of proteins in the chromatin of ovary sections strongly decreases the accessibility of Sl endonuclease to DNA; this explains why one must use prolonged times of incubation and high concentrations of the enzyme in order to follow its effects in situ, as we have tried to do. Although the morphology of the “caps” which are very compact structures, does not seem affected by the enzymic treatment, without previous thermal denaturation, the [3H]TdR labelled rDNA which they contain is hydrolysed at a temperature (37°C) where Sl endonuclease is
441
almost ineffective upon double-stranded DNA. Its action is detectable on doublestranded DNA only after thermal denaturation at 100°C [ll]. Under such conditions (after heating at lOO”C), besides “cap” DNA, the follicular cell nuclei are affected by the enzyme. Our results, in this respect, confirm those of Dreskin & Mayall [ 1l] who studied the effects of thermal denaturation on human leukocyte chromatin in situ with a different staining method (gallocyanin chromalun). Besides the fact that rapid replication takes place in rDNA, our present in situ observations on the effects of Sl endonuclease from Neurospora crassa confir~-~~ that single-stranded forms are present in “cap” DNA, that they are labelled with r3H]TdR and that they could play an intermediary role in the gene amplification process. We wish to thank Dr Christopher Evans for kind help in the preparation of the manuscript.
References 1. Gall,JG&Pardue,ML,ProcnatlacadsciUS63 (1%9) 378. 2. MacGregor, H C, J cell sci 3 (1%8) 437. 3. Ficq, A, Exp cell res 53 (1968) 691. 4. -Ibid 63 (1970) 453. 5. Durand, M, Compt rend acad sci 234 (1952) 1579. 6. Thomas, R, Arch int physio161 (1953) 370. 7. Buongiomo-Nardelli, M, Amaldi, F & LavaSanchez, PA, Exp cell res 98 (1976) 95. 8. Hourcade, D, Dressler, D & Wolfson, J, Proc natl acad sci US 70 (1973) 2926. 9. Rochaix, J B, Bird, A & Bakken, A, J mol biol 87 (1974) 473. 10. Gall, J G, Symposium on the nucleolus. Natl cancer inst monogr 23 (1966) 475. 11. Dreskin, S C & Mayall, B H, J histochem cyto-. them 22 (1974) 120. 12. Gall. J G. Proc natl acad sci US 60 (1968) 533. 13. Ficq; A, J microsc biol cell 25 (1976) 7. ’ 14. - The cell (ed J Brachet & A Mirsky) vol. I, p. 67. Academic Press, New York (1959). Received September 21, 1976 Accepted October 21, 1976
Exp Cell Res 104 (1977)